Cancer cells are characterized by their ability to proliferate in a continuous, uncontrolled fashion. It is now clear that primary cell cycle regulators must be circumvented or directly involved in oncogenesis in order for this to occur.
Cell cycle regulation occurs at the boundaries of the G.sub.1 /S and G.sub.2 /M phases, two major transition points of the cell cycle. A key regulator of these transitions is p34.sup.cdc2 kinase which is known to phosphorylate a number of proteins including histone H1, DNA polymerase .alpha., RNA polymerase II, retinoblastoma tumor suppressor protein (Rb), p53, nucleolin, cAb1, SV40 large T antigen and lamin A. For example, p34.sup.cdc2 kinase activity is required for entry of cells into mitosis, i.e., for passage from the G.sub.2 phase of the cell cycle into the M phase. (Lee et al. (1988) Trends Genet. 4:289-90; Dunphy et al. (1988) Cell 54:423-431; Gautier et al. (1988) Cell 54:433-439; see, for review, Cross et al. (1989) Ann. Rev. Cell Biol. 5:341-395; Hunt (1989) Curr. Opinion Cell Biol. 1:268-274; Nurse (1990) Nature 344:503-508).
The activity of p34.sup.cdc2 kinase is, in turn, regulated by both protein:protein interactions and post-translational modifications. Thus, blockage of either of these mechanisms leads to arrest of the mammalian cell cycle. For example, microinjection of p34.sup.cdc2 antibodies into serum-stimulated rat fibroblasts causes cells to arrest in G.sub.2 and treatment of activated T lymphocytes with p34.sup.cdc2 anti-sense oligodeoxynucleotides inhibits DNA synthesis (Furukawa et al. (1990) Science 250:805-808; Riabowol et al. (1989) Cell 57:393-401). Injection of suc1 protein (a ligand of p34.sup.cdc2) into HeLa cells arrests cell growth, presumably by disrupting normal p34.sup.cdc2 protein:protein interactions (Draetta (1990) TIBS 15:378-383). In addition, inhibition of the cdc25 phosphatase with specific antibodies blocks the post-translational modification of p34.sup.cdc2 and leads to HeLa cell death (Galaktionov et al. (1991) Cell 67:1181-1194).
Rb, p107 protein and the cyclin (cyc) protein family have been shown to associate with p34.sup.cdc2 (and its homolog p33.sup.cdk2) (Pines et al. (1990) Nature 346:760-763; Tsai et al. (1991) Nature 353:174-177; Giordano et al. (1989) Cell 58:981-990); the E2F transcription factor; the adenovirus E1A protein; and the human papillomavirus transforming protein E7 (Whyte et al. (1988) Nature 334:124-129; Chelappan et al. (1991) Cell 65:1053-1061; Bandara et al. (1991) Nature 352:249-252; Mundryj et al. (1991) Cell 65:1243-1253; Pines et al. (1990), supra; Tsai et al. (1991), supra; Giordano et al. (1989), supra; Shirodkar et al. (1992) Cell 68:157-166; Devoto et al. (1992) Cell 68:157-166; DeCaprio et al. (1988) Cell 54:275-283; Dyson et al. (1989) Science 243:934-937; Gage et al. (1990) J. Virol. 64:723-730). The binding of cyclins to p34.sup.cdc2 or p33.sup.cdk2 is required for kinase activity (Solomon et al. (1990) Cell 63:1013-1024; Pines et al. (1990), supra; Tsai et al. (1991), supra; Giordano et al. (1989), supra).
The functional domains of the Rb and p107 proteins have been mapped through both genetic and biochemical means. (Hu et al. (1990) EMBO J. 9:1147-1155; Ewen et al. (1991) Cell 66:1155-1164; Ewen et al. (1992) Science 255:85-87). An approximately 400 amino acid fragment of Rb and p107, termed the Rb pocket, is responsible for association of these proteins with the DNA tumor virus oncoproteins and cellular ligands. Within this domain are six regions of extensive sequence similarity between Rb and p107. (Ewen et al. (1991), supra). Likewise, the cyclins share a large region of sequence similarity spanning approximately 87 amino acids, which has been designated the "cyclin box." (Pines et al. (1989) Cell 58:833-846) This domain is thought to play a role in protein:protein interactions and it has been shown that deletion of sequences amino-terminal to this domain do not affect cyclin function. (Murray et al. (1989) Nature 339:280-286; Lew et al. (1991) Cell 66:1197-1206).
p34.sup.cdc2 protein:protein interactions are altered in human tumors. For example, the gene encoding the cofactor cyclin A is disrupted in hepatocellular carcinoma (Wang et al. (1990) Nature 343:555-557). Also, recent data have demonstrated that cyclin D1 (PRAD1) is within the bc1-1 locus and is rearranged in parathyroid tumors and some B cell leukemias (de Boer et al. (1993) Cancer Res. 53:4148-4152; Motokura et al. (1991) Nature 350:512-515). In addition, the bc1-1 locus is frequently amplified in breast carcinoma and cyclin D1 is overexpressed in mouse skin carcinoma (Lammie et al. (1991) Oncogene 6:439-444; Bianchi et al. (1993) Oncogene 8:1127-1133; Buckley et al. (1993) Oncogene 8:2127-2133). Furthermore, the subunits of the cdk kinases are rearranged in transformed cells when compared to their normal counterpart (Xiong et al. (1993) Genes and Development 7:1572-1583). This is the result of the loss or underexpression of waf1/cip1 protein, which is normally a repressor of cdk kinase activities, and is regulated by the p53 tumor suppressor protein (Xiong et al. (1993) Nature 366:701-704; Serrano et al. (1993) Nature 366:704-707; Gu et al. (1993) Nature 366:707-710; Harper et al. (1993) Cell 75:805-816; El-Deiry et al. (1993) Cell 75:817-825). These data clearly implicate the alteration of p34.sup.cdc2 kinase activity in oncogenesis.
Accordingly, inhibitors of p34.sup.cdc2 activity would be useful in the regulation and control of the continuous, proliferative growth of cancerous cells.